Targeting epigenetic repression by interfering with methyl-binding domain protein function

Hypermethylation of CpG islands is a common method for silencing tumor suppressor genes in cancer. Combating this, drugs have targeted both the methylation maintenance enzymes (DNA methyltransferases) and histone deacetylases which create the repressed chromatin architecture. Here, we present data on two methods to target this pathway. The first relies on inhibiting the interaction between methyl-binding domain proteins and C5mepG's within DNA. This new compound class was discovered and characterized by high throughput screening, RTPCR and biochemical assays resulting in both methyl-binding protein specific and nonspecific inhibitors. They all synergize with the known methyltransferase inhibitor 5-aza-2'-deoxycytidine. We even show preclinical efficacy with one of them by sensitizing a retinoid resistant cell line in culture and xenografted mice. Results from this study also led us to determine that MBD2 is phosphorylated and loss of Aurora kinase A reactivates the epigenetically silenced gene pi-class glutathione S-transferase 1. Then, we further characterized the mechanism of how the halogenated uridine 5-bromo-2'-deoxyuridine can activate hypermethylated genes. Previous studies have described changing transcription factor:DNA affinity, while others have shown no incorporation at the immediate promoter of activated genes. Our data supports the need for low levels of incorporation locally to activate genes, with no incorporation density effect. We theorize this mechanism relies on histone's decreased affinity for DNA substituted with a minimal number of halogenated bases.In summary, we have discovered new small molecule inhibitors to epigenetic repression that work by the novel mechanism of inhibiting a transcription repressor:DNA binding event, while further exploring the incorporation dependent mechanism of halogenated uridines.